Research ArticleHeart Failure

Central-acting therapeutics alleviate respiratory weakness caused by heart failure–induced ventilatory overdrive

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Science Translational Medicine  17 May 2017:
Vol. 9, Issue 390, eaag1303
DOI: 10.1126/scitranslmed.aag1303
  • Fig. 1. End-stage pressure overload HF induces diaphragmatic myopathy, elevated ventilatory drive, and pulmonary remodeling in the absence of pulmonary edema.

    (A) Point of constriction on the transverse and abdominal aorta for experimental cardiac pressure overload. (B) Representative cross-sectional images of sham and 18-week TAC hearts at the mid-papillary region and stained with picrosirius red. (C) Normalized cardiac weights of whole heart (WH), left ventricle (LV), and right ventricle (RV) of sham (n = 8) and 18-week TAC (n = 8) mice. (D and E) The first derivative of pressure development (D) and left ventricular pressure (E) in sham (n = 8) and 18-week TAC (n = 8) mice. EDP, end-diastolic pressure. (F) In vivo maximal inspiratory pressure of sham (n = 10) and 18-week TAC (n = 6) mice during a 25-s airway occlusion. (G) In vitro force production of diaphragm from sham (n = 12) and 18-week TAC (n = 10) mice. (H) Duty cycle of sham (n = 8) and 18-week TAC (n = 5) mice. (I) Representative images of sham and 18-week TAC diaphragms stained with picrosirius red. (J) Cross-sectional area (CSA) of diaphragm muscle fibers in sham (n = 5) and 18-week TAC (n = 5) mice. (K) Percent fibrosis of sham (n = 5) and 18-week TAC (n = 5) diaphragms. (L) Twitch and tetanic diaphragmatic forces for sham (n = 5) and 18-week TAC (n = 5) mice. (M) Whole lungs of sham and 18-week TAC mice perfusion fixed at 20 cmH2O and stained with Gomori’s trichrome. (N) Wet weight–to–dry weight ratio of lungs from sham (n = 8) and 18-week TAC (n = 8) mice. (O) Wet and dry weights of sham (n = 8) and 18-week TAC (n = 8) lungs. Mean ± SEM; *Significance versus sham; P < 0.05, as determined by Student’s t test. Scale bars, 2 mm (B), 100 μm (I), and 100 μm (M).

  • Fig. 2. Diaphragmatic myopathy progressively develops throughout the duration of 18 weeks of TAC and is characterized by atrophy and decreased PIocc.

    (A) Representative tracings of inspiratory pressure at baseline during anesthetized eupneic breathing (PI) and maximum inspiratory pressure (PIocc) during a 25-s airway occlusion. (B) Representative tracings of inspiratory pressure development during airway occlusion in TAC and sham mice. (C) Inspiratory pressure development in sham (n = 10) and TAC (n ≥ 6) mice during the 25-s airway occlusion. (D) Representative images of sham and TAC diaphragms stained with hematoxylin and eosin (H&E) and wheat germ (WGA) demonstrating diaphragm atrophy during the progression of TAC. (E) Diaphragm muscle fiber CSA histogram of sham (n ≥ 400 technical replicates for each of the five biological replicates) versus TAC (n ≥ 400 technical replicates for each of the five biological replicates in each group) mice. (F) Correlation between PIocc and diaphragm muscle fiber CSA of sham (n = 10) and TAC (n ≥ 6) mice. Mean ± SEM; *Significance versus sham; P < 0.05, as determined by protected least-squares difference (LSD). Scale bars, 10 cmH2O (vertical bar) and 5 s (horizontal bar) (A and B) and 240 μm (top) and 35 μm (bottom) (D).

  • Fig. 3. TAC induces progressive and profound lung remodeling that reduces dynamic lung compliance in late-stage TAC.

    (A) Lung wet and dry weights of sham and TAC mice. (B) Representative images of lung tissue stained with Gomori’s trichrome. (C) Interstitial/alveolar pulmonary fibrosis (n = 5). (D) Histogram of alveolar CSA in sham (n = 5) and TAC (n = 5) mice. (E) Representative raw pressure tracings during 200 μl of lung inflation for the measurement of dynamic lung compliance in sham and TAC mice. (F) Dynamic lung compliance curves of sham (n = 4) and TAC (n = 4 for each group) mice. Mean ± SEM; *Significance versus sham; P < 0.05, as determined by protected LSD. Scale bars, 30 μm (top) and 15 μm (bottom) (B).

  • Fig. 4. ANGII and β-ADR codependent signaling stimulates neural ventilatory drive that is only normalized by antagonists that cross the BBB.

    (A) Representative tracings of inspiratory pressure during anesthetized breathing at rest in sham and TAC mice. (B) Inspiratory pressure (PI) and (C) ventilatory drive (PI/TI) during anesthetized breathing in sham and TAC mice. (D) Inspiratory pressure of sham and TAC mice following acute treatment with captopril or propranolol. (E) Inspiratory pressure measured during the infusion of ANGII or isoproterenol in the presence or absence of captopril or propranolol (n = 4 for each combination). (F) Representative tracing of diaphragmatic EMG and airway pressure during the infusion of ANGII. (G) Inspiratory pressure in sham and 2-week TAC mice in the presence or absence of ANGII or β-ADR receptor blockers (n = 8 for each combination). (H) Chart indicating the ability of various ANGII and β-ADR receptor blockers to cross the BBB. Mean ± SEM; *Significance versus sham; P < 0.05, as determined by protected LSD. Carvediol is rapidly effluxed and does not accumulate within the brain.

  • Fig. 5. Chronic β-ADR blockade normalizes diaphragm in vitro function that is not explained by acute β-ADR stimulation or alterations in myofilament function.

    (A) Raw tracings of in vitro tetanic contraction from sham, sham + isoproterenol (ISO), and 2-week TAC diaphragm. (B) Maximal in vitro force production of sham (n = 10), sham + isoproterenol (n = 6), and 2-week TAC diaphragm (n = 14). (C) Diaphragm myofilament force production and (D) calcium sensitivity (pCA50) of sham (n = 17) and 2-week TAC (n = 19) mice. (E) Relative mRNA expression in sham, 2-week TAC, and 2-week TAC + propranolol mice (n = 3 technical replicates for each of the five biological replicates in each group). (F) Peak force production and (G) maximum rate of force production +dT/dtmax during force-frequency protocol in sham (n = 12), 2-week TAC (n = 14), and 2-week TAC + propranolol (n = 7) mice. Representative tracings of in vitro fatigue protocol in (H) 2-week TAC and (I) 2-week TAC + propranolol diaphragms. (J) Maximum passive force development of sham (n = 12), 2-week TAC (n = 14), and 2-week TAC + propranolol (n = 7) diaphragms during in vitro fatiguing stimulations. All mRNAs are expressed relative to the housekeeping gene Hmbs (hydroxymethylbilane synthase). Mean ± SEM; *Significance versus sham; P < 0.05, as determined by protected LSD. #Significance versus 2-week TAC + propranolol; P < 0.05, as determined by protected LSD.

  • Fig. 6. BBB-permeant β-blockers ameliorate diaphragm atrophy, restore inspiratory strength, and normalize EIF2α phosphorylation and relative Perk expression in TAC mice.

    (A) Representative cross-sectional images of diaphragm from sham, 4-week TAC, 4-week TAC + propranolol (pro), and 4-week TAC + atenolol (ateno) stained with picrosirius red. (B) Diaphragm muscle fiber CSA histogram of sham (n ≥ 400 technical replicates for each of the five biological replicates) versus 4-week TAC + propranolol mice (n ≥ 400 technical replicates for each of the five biological replicates) or (C) 4-week TAC + atenolol mice (n ≥ 400 technical replicates for each of the five biological replicates). (D) Maximal inspiratory pressure during a 25-s airway occlusion in sham (n = 10), 4-week TAC (n = 8), 4-week TAC + propranolol (n = 6), and 4-week TAC + atenolol (n = 5) mice. (E to I) Relative diaphragm mRNA expression of key gene markers involved in (E) apoptosis, (F) autophagy, (G) degradation, (H) regeneration, and (I) the unfolded protein response in sham and 4-week TAC mice (n = 3 technical replicates for each of the five biological replicates in each group). (J) Relative diaphragm Perk mRNA expression in sham, 4-week TAC, 4-week TAC + propranolol, or atenolol-treated mice (n = 3 technical replicates for each of the five biological replicates in each group). (K) Diaphragm total protein content, (L) phosphorylation, and (M) protein/phosphorylation ratio of sham (n = 5), 4-week TAC (n = 5), and 4-week TAC + propranolol (n = 5) with MemCode stains and Western blots. All mRNA are expressed relative to the housekeeping gene Hmbs. Mean ± SEM; *Significance versus sham. #Significance versus 4-week TAC; P < 0.05, as determined by protected LSD. Scale bar, 50 μm (A).

  • Fig. 7. Heart failure–induced ventilatory overdrive triggers the development of diaphragm atrophy and weakness independent of pulmonary edema or lung remodeling.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/390/eaag1303/DC1

    Fig. S1. TAC induces progressive interstitial fibrosis in the diaphragm.

    Fig. S2. TAC induces biphasic and transient increases in diaphragm in vitro–specific force production, contractility, and passive baseline force.

    Fig. S3. AAC induces diaphragmatic atrophy and fibrosis.

    Fig. S4. Peripheral limb muscle CSA is unaffected in mice at 4 weeks of TAC.

    Fig. S5. Propranolol and atenolol treatment in sham mice induces alterations in diaphragm gene expression profile.

    Fig. S6. Propranolol and atenolol treatment in TAC mice induces alterations in diaphragm gene expression profile.

    Table S1. TAC induces progressive changes in hemodynamics, morphometrics, and metabolic parameters throughout 18 weeks of pressure overload.

    Table S2. Pressure overload imposed by 18 weeks of AAC induces hemodynamic and morphometric alterations similar to 9 weeks of TAC.

    Table S3. Indices of systolic and diastolic dysfunction persist in 2-week and 4-week TAC mice treated with propranolol.

  • Supplementary Material for:

    Central-acting therapeutics alleviate respiratory weakness caused by heart failure–induced ventilatory overdrive

    Andrew J. Foster, Mathew J. Platt, Jason S. Huber, Ashley L. Eadie, Alicia M. Arkell, Nadya Romanova, David C. Wright, Todd E. Gillis, Coral L. Murrant, Keith R. Brunt,* Jeremy A. Simpson*

    *Corresponding author. Email: jeremys{at}uoguelph.ca (J.A.S.); keith.brunt{at}dal.ca (K.R.B.)

    Published 17 May 2017, Sci. Transl. Med. 9, eaag1303 (2017)
    DOI: 110.1126/scitranslmed.aag1303

    This PDF file includes:

    • Fig. S1. TAC induces progressive interstitial fibrosis in the diaphragm.
    • Fig. S2. TAC induces biphasic and transient increases in diaphragm in vitro–specific force production, contractility, and passive baseline force.
    • Fig. S3. AAC induces diaphragmatic atrophy and fibrosis.
    • Fig. S4. Peripheral limb muscle CSA is unaffected in mice at 4 weeks of TAC.
    • Fig. S5. Propranolol and atenolol treatment in sham mice induces alterations in diaphragm gene expression profile.
    • Fig. S6. Propranolol and atenolol treatment in TAC mice induces alterations in diaphragm gene expression profile.
    • Table S1. TAC induces progressive changes in hemodynamics, morphometrics, and metabolic parameters throughout 18 weeks of pressure overload.
    • Table S2. Pressure overload imposed by 18 weeks of AAC induces hemodynamic and morphometric alterations similar to 9 weeks of TAC.
    • Table S3. Indices of systolic and diastolic dysfunction persist in 2-week and 4-week TAC mice treated with propranolol.

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